Activators of ATP-gated ion channels (P2X receptors) are being synthesized and investigated for cardioprotection in collaboration with Dr. Bruce Liang (University of Connecticut). MRS2339, synthesized in our lab, is a nucleotide activator of a P2X4R ion channel present in the cardiac muscle cells. We have explored the structure activity relationships of this nucleotide. Certain phosphonate derivatives are more stable to hydrolysis than the phosphate derivative MRS2339 and are being explored in vivo. MRS2339 is currently being licensed by private industry for the treatment of heart failure. The P2X ion channels mediate a number of potent and possibly important biological effects in the cardiovascular, inflammatory, and central nervous systems. Previous studies have shown that extracellular ATP can cause an ionic current in murine, rat and guinea pig cardiac ventricular myocytes. The receptor that mediates this current appears to be a P2X receptor, of which the P2X4 receptor is an important subunit. Activation of P2X receptors leads to the opening of a nonselective cation channel permeable to sodium, potassium, and calcium ions. The current is inward at negative membrane potentials, reverses near 0 mV, and becomes outward at positive potentials. The continuous activation of this receptor channel by endogenous extracellular ATP may assume an important biological function. This constant activation under the resting or negative membrane potentials would produce an inward current, whereas its activation during depolarized portions of the action potential should lead to an outward current. These currents represent a possible ionic mechanism by which the cardiac P2X channel achieves its biological effects. A potential biologically important role of the cardiac P2X receptor was suggested by the finding that cardiac myocyte-specific overexpression of the P2X4 receptor can rescue the hypertrophic and heart failure phenotype of the calsequestrin (CSQ) model of cardiomyopathy. However, little is known regarding regulation of the cardiac P2X receptor in cardiac hypertrophy or failure. Furthermore, it is not clear whether an increased activation of the endogenous P2X receptor channel is beneficial or harmful in the progression of heart failure. The regulation of the P2X receptor-mediated ionic current and its potential role in heart failure was investigated using several novel nucleotide agonists. Chronic administration of a novel nucleotidase-resistant P2 receptor agonist MRS2339, which was capable of inducing this ionic current and was devoid of any vasodilator action, reduced cardiac hypertrophy and increased lifespan. The data suggests that an important biological function of the cardiac P2X current is to favorably modulate the progression of cardiac hypertrophy and failure. Recently we identified uncharged carbocyclic nucleotide analogues (including nonhydrolyzable phosphonates) related structurally to MRS2339, that represent potential candidates for the treatment of heart failure, suggesting this as a viable and structurally broad approach. We also found a beneficial therapeutic effect of 2-cyclohexylthio-adenosine 5-monophosphate in mice with heart failure (HF). Heart failure (HF), despite continuing progress, remains a leading cause of mortality and morbidity. P2X4 receptors (P2X4R) have emerged as potentially important molecules in regulating cardiac function and as potential targets for HF therapy. Transgenic (Tg) P2X4R overexpression can protect against HF. In collaboration with Bruce T. Liang, we have now defined the role of native cardiac P2X4R under basal conditions and during HF induced by myocardial infarction or pressure overload. Mice established with a conditional cardiac-specific P2X4R knockout (KO) were subjected to left coronary ligation-induced post-infarct or transverse aorta constriction-induced pressure overload HF. KO cardiac myocytes did not show P2X4R by immunoblotting or by any response to the P2X4R-specific allosteric enhancer ivermectin. KO hearts showed normal basal cardiac function but depressed contractile performance in post-infarct and pressure overload models of HF by in vivo echocardiography and ex vivo isolated working heart parameters. P2X4R co-immunoprecipitated and co-localized with nitric oxide synthase 3 (eNOS) in wild type cardiac myocytes. Mice with cardiac-specific P2X4R overexpression had increased S-nitrosylation, cGMP, NO formation, and were protected from post infarct and pressure overload HF. Inhibitor of eNOS L-NIO blocked the salutary effect of cardiac P2X4R overexpression in post-infarct and pressure overload HF as did eNOS knockout. This study established a new protective role for endogenous cardiac myocyte P2X4R in HF and demonstrated a physical interaction between the myocyte receptor and eNOS, a mediator of HF protection. We have discovered the first allosteric modulators of the dopamine transporter (DAT) in collaboration with Aaron Janowsky. These rigidified nucleoside derivatives inhibited dopamine uptake, but enhanced the binding of a tropane radioligand to the transporter. The most potent analogue displayed an EC50 value of 35 nM in the enhancement of radioligand binding. in some cases, in some cases modulation of the norepinephrine transporter(NET) was also observed. The behavioral effects of these agents are being probed. These allosteric ligands stability the human form of DAT such that it can be purified to homogeneity for the first time (with Eric Gouaux). We have provided compounds for the study of transient receptor potential cation channel subfamily V member 1 (TRPV1) to our collaborator Dr. Michael Iadarola of NIH. TRPV1 functions as a multimodal nociceptor gated by high temperatures, protons, and small molecule vanilloid ligands such as capsaicin. The ability to respond to a variety of stimuli (heat, low pH, vanilloids, and endovanilloids) and its altered sensitivity and expression in experimental inflammatory and neuropathic pain models made TRPV1 a major target for the development of novel, nonopioid analgesics. These have been mostly antagonists, which have intolerable side effects in human clinical trials, but recent work shows that potent agonists or enhancers agonists have utility in this context. Here we show that the dihydropyridine derivative 4,5-diethyl-3-(2-methoxyethylthio)-2-methyl-6-phenyl-1,4-dihydropyridine-3,5-dicarboxylate (MRS1477) behaves as a positive allosteric modulator of both proton and vanilloid activation of TRPV1. Under inflammatory mimetic conditions of low pH (6.0) and protein kinase C phosphorylation, addition of MRS1477 further increased sensitivity of already sensitized TPRV1 toward capsaicin. MRS1477 does not affect inhibition by known vanilloid antagonists and remains effective in potentiating activation by pH in the presence of an orthosteric vanilloid antagonist. These results indicate a distinct site on TRPV1 for positive allosteric modulation that may bind endogenous compounds or novel pharmacological agents. Positive modulation of TRPV1 sensitivity suggests that it may be possible to produce a selective analgesia through calcium overload restricted to highly active nociceptive nerve endings at sites of tissue damage and inflammation.